Coronavirus modulation of cellular microRNAs in the liver: role in hepatitis

冠状病毒对肝脏细胞 microRNA 的调节：在肝炎中的作用

• 批准号: 8113557
• 负责人: SONIA NAVAS-MARTIN
• 金额: $ 23.18万
• 依托单位: DREXEL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-29 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8113557
• 关键词: Acute; Acute Hepatitis; Address; Animals; Binding Sites; Biological; Biological Assay; Cell physiology; Cells; Complex; Computer Simulation; Coronavirus; Coronavirus Infections; DNA Viruses; Data; Disease; Disease Outcome; Elements; Endothelial Cells; Experimental Models; Functional RNA; Gene Expression; Genes; Genome; Health; Hepatic; Hepatitis; Hepatitis B; Hepatitis C; Hepatocyte; Human; Immune system; In Vitro; Infection; Inflammation; Inflammatory; Interferon Type II; Interferons; Intervention; Investigation; Knowledge; Kupffer Cells; Liver; Liver diseases; Luciferases; Mediating; Messenger RNA; MicroRNAs; Microarray Analysis; Modeling; Molecular; Molecular Profiling; Murine hepatitis virus; Mus; Nuclear; Outcome; Pathogenesis; Pattern; Platelet Factor 4; Play; Poly(A)+ RNA; RNA Viruses; Regulation; Regulatory T-Lymphocyte; Relative (related person); Reporter; Reverse Transcriptase Polymerase Chain Reaction; Role; Small RNA; System; Testing; Therapeutic; Time; Translational Repression; Translations; Tropism; Untranslated Regions; Validation; Viral; Viral Genes; Viral hepatitis; Virus; Virus Diseases; base; cellular targeting; cytokine; human coronavirus; in vivo; innovation; knock-down; liver function; mRNA Transcript Degradation; macrophage; man; novel; pathogen; positional cloning; promoter; prototype; respiratory; response; transcription factor; virus host interaction

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are small RNAs that regulate gene expression by translational repression and/or mRNA degradation. miRNAs may also up-regulate translation under certain circumstances. There is increasing evidence indicating that miRNAs play a major role in fundamental cellular processes, the regulation of the immune system, as well as in the onset and progression of many diseases. The role of miRNAs in virus-host interaction is just starting to be addressed. miRNAs have been found in some viruses and miRNAs from the host cell may play a role in regulating viral genes and even determine viral tropism. We have investigated whether coronaviruses (CoVs), a group of emerging animal and human RNA viruses, may have the ability to modulate the expression of some cellular miRNAs, which may determine disease outcome. Using a reverse genetic system to manipulate the CoV genome, we have previously characterized the molecular determinants of CoV-induced hepatitis, showed that the ability of murine coronaviruses to induce hepatitis is virus strain specific, and identified cellular targets in the liver (hepatocytes, endothelial cells, and the resident macrophage Kupffer cells). In Preliminary Studies, we have used miRNA microarray analysis and validation by real-time RTPCR to provide evidence of the differential expression of selected cellular miRNAs in macrophages upon in vitro infection with various strains of murine CoVs that induce different hepatitis outcome in the mouse (from minimal inflammation to fulminant hepatitis). Changes in the macrophage miRNAs profiles after murine CoVs infection were overlapping but distinct, and were observed early after infection in the absence of type I IFNs, IFN-?, and pro-inflammatory cytokine secretion. Therefore, we hypothesized that early alteration of the miRNAs expression profile in the CoV-infected mouse liver is a consequence of virus infection, and that strain-specific changes in miRNAs expression contribute to the differences in hepatitis outcome. Thus, we propose the following Specific Aims: (1) Using microarray analysis and real time RT-PCR, we will determine alterations in miRNA profiles in the mouse liver and in primary liver cells (hepatocytes and Kupffer cells) freshly isolated from infected mice, which are different between murine CoVs that differ in hepatitis outcome (MHV-A59, acute moderate hepatitis; and MHV-3, fulminant hepatitis); (2) Based upon results in Specific Aim 1, we will subsequently identify potential cellular miRNAs targets in 3'UTRs by in silico and functional approaches, focusing on those miRNAs whose expression is differentially modulated (up or down) between the viruses listed above and might be involved in fulminant hepatitis; and, (3) We will define the biological effects of knocking-down selected miRNAs (identified in the previous aims) in Kupffer and regulatory T cells and determine the role that those selected miRNAs may have in CoV-induced hepatitis outcome by specific in vivo miRNA-silencing using antagomirs.

描述（由申请人提供）：MicroRNA (miRNA) 是通过翻译抑制和/或 mRNA 降解调节基因表达的小 RNA。在某些情况下，miRNA 也可能上调翻译。越来越多的证据表明 miRNA 在基本细胞过程、免疫系统调节以及许多疾病的发生和进展中发挥着重要作用。 miRNA 在病毒与宿主相互作用中的作用才刚刚开始得到解决。在一些病毒中发现了 miRNA，来自宿主细胞的 miRNA 可能在调节病毒基因甚至决定病毒趋向性方面发挥作用。我们研究了冠状病毒 (CoV)（一组新兴的动物和人类 RNA 病毒）是否具有调节某些细胞 miRNA 表达的能力，这可能决定疾病的结果。利用反向遗传系统操纵 CoV 基因组，我们之前已经表征了 CoV 诱发肝炎的分子决​​定因素，表明鼠冠状病毒诱发肝炎的能力是病毒株特异性的，并确定了肝脏中的细胞靶点（肝细胞、内皮细胞）。细胞和常驻巨噬细胞库普弗细胞）。在初步研究中，我们使用 miRNA 微阵列分析和实时 RTPCR 验证来提供巨噬细胞中选定的细胞 miRNA 在体外感染各种鼠科冠状病毒菌株后差异表达的证据，这些菌株在小鼠中诱导不同的肝炎结果（来自轻微炎症至暴发性肝炎）。小鼠 CoV 感染后巨噬细胞 miRNA 谱的变化是重叠但又不同的，并且在感染后早期在 I 型 IFN、IFN-γ 和促炎细胞因子分泌不存在的情况下观察到。因此，我们假设 CoV 感染的小鼠肝脏中 miRNA 表达谱的早期改变是病毒感染的结果，并且 miRNA 表达的菌株特异性变化导致肝炎结果的差异。因此，我们提出以下具体目标：（1）使用微阵列分析和实时RT-PCR，我们将确定小鼠肝脏和从感染小鼠中新鲜分离的原代肝细胞（肝细胞和库普弗细胞）中miRNA谱的变化，不同肝炎结局的小鼠 CoV 之间存在差异（MHV-A59，急性中度肝炎；MHV-3，暴发性肝炎）； (2) 基于具体目标 1 中的结果，我们随后将通过计算机和功能方法识别 3'UTR 中潜在的细胞 miRNA 靶标，重点关注那些在上述病毒和病毒之间表达受到差异调节（上调或下调）的 miRNA。可能涉及暴发性肝炎； (3) 我们将定义敲低 Kupffer 细胞和调节性 T 细胞中选定的 miRNA（在之前的目标中确定）的生物学效应，并通过特定的体内试验确定这些选定的 miRNA 在 CoV 诱导的肝炎结果中可能发挥的作用使用 antagomir 进行 miRNA 沉默。